2.2 Reagents
and Equipment
- 300–500μM Dichlorophenolindophenol (DCPIP) stock solu-
tion (10) in nanopure water. - 3–10 mM SAM stock solution (10) in nanopure water.
- 100μl Cuvettes or 96/384-well plates (seeNote 3).
- Acyl-CoA stocks (5–20) in nanopure water to cover a range
of 1–200 μM final acyl-CoA concentration in the assay
(seeNote 4). - 1 M HEPES buffer pH 7.3 (10).
- 1–10 μM Stock Bacillus subtilis Sfp phosphopantetheinyl
transferase. - 5μM StockEscherichia colimethylthioadenosine nucleosidase
(MTAN). - 20 units/ml xanthine oxidase.
- Other reagents: Glycerol, 10 mM 2-(N-morpholino)ethane-
sulfonic acid (MES), 0.5 M potassium chloride, acetic acid,
HPLC-grade acetonitrile, HPLC-grade methanol, 2 M sodium
acetate pH 4, trifluoroacetic acid (TFA). - UV-visible spectrophotometer.
- HPLC instrument with autosampler and PDA detector.
Fig. 4Coupled assay. (a) Assay principle (b) Background reaction progress curve for 300μM SAM, 50μM
C4-ACP, 0.5μM MTAN, and 2 units/ml xanthine oxidase. (c) Enzyme reaction progress curve. The assay
mixture includes all components for background curve described in (b) and 0.3μM RhlI
Acyl-Homoserine Lactone Synthase Assays 165